Plasma and cerebrospinal fluid pharmacokinetics of vodobatinib, a neuroprotective c-Abl tyrosine kinase inhibitor for the treatment of Parkinson's disease.

BACKGROUND: Preclinical evidence suggests that c-Abl is critical in the pathogenesis of Parkinson's Disease (PD). Vodobatinib (K0706) is a potent, specific Abl kinase inhibitor currently being developed for the treatment of PD. In previously reported studies, nilotinib, a multikinase c-Abl inhibitor, did not show clinical activity as evidenced by no improvement of symptoms or the rate of decline after one to six months of treatment at the maximum permissible dose, presumably because of insufficient CNS penetration. Here we report clinical PK and safety data for vodobatinib. OBJECTIVES: To determine safety, plasma PK, and CSF penetration of vodobatinib in healthy volunteers and PD subjects following oral administration, and compare CSF levels to in vitro concentrations required for c-Abl inhibition relative to data reported for nilotinib. METHODS: Inhibition of c-Abl kinase activity and c-Abl binding affinity were first assessed in vitro. Healthy human volunteers and PD patients received various oral doses of vodobatinib once-daily for seven and fourteen days respectively, to assess safety, and plasma and CSF PK. RESULTS: In in vitro assays, vodobatinib was more potent (kinase IC50 = 0.9 nM) than nilotinib (kinase IC50 = 15-45 nM). Administration of vodobatinib 48, 192 and 384 mg to healthy subjects for 7 days yielded mean Cmax, CSF values of 1.8, 11.6, and 12.2 nM respectively, with the two highest doses exceeding the IC50 over the entire dosing interval. Cavg, CSF values were 6-8 times greater than the IC50. Comparable CSF levels were observed in PD patients. All doses were well tolerated in both cohorts. CONCLUSION: Based on achieved CSF concentrations, the potential for c-Abl inhibition in the brain is substantially higher with vodobatinib than with nilotinib. The CSF PK profile of vodobatinib is suitable for determining if c-Abl inhibition will be neuroprotective in PD patients.

ASN004, A 5T4-targeting scFv-Fc Antibody-Drug Conjugate with High Drug-to-Antibody Ratio, Induces Complete and Durable Tumor Regressions in Preclinical Models.

The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors but shows very limited expression in normal adult tissues. ASN004 is a 5T4-targeted antibody-drug conjugate (ADC) that incorporates a novel single-chain Fv-Fc antibody and Dolaflexin drug-linker technology, with an Auristatin F hydroxypropylamide payload drug-to-antibody ratio of approximately 10-12. The pharmacology, toxicology, and pharmacokinetic properties of ASN004 and its components were investigated in vitro and in vivo ASN004 showed high affinity for the 5T4 antigen and was selectively bound to and internalized into 5T4-expressing tumor cells, and potent cytotoxicity was demonstrated for a diverse panel of solid tumor cell lines. ASN004 induced complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a wide range of 5T4 expression levels. A single dose of ASN004, as low as 1 mg/kg i.v., achieved complete tumor regression leading to tumor-free survivors in the A431 cervical cancer model. In head-to-head studies, superior activity of ASN004 was demonstrated against trastuzumab-DM1, in a low-5T4/high-HER2 expressing gastric tumor model, and 10-fold greater potency was found for ASN004 against the 5T4-targeted ADC PF-06263507 in a lung tumor model. In marmoset monkeys, ASN004 was well tolerated at doses up to 1.5 mg/kg Q3W i.v., and showed dose-dependent exposure, linear pharmacokinetics, and markedly low exposure of free payload drug. Taken together, these findings identify ASN004 as a promising new ADC therapeutic for clinical evaluation in a broad range of solid tumor types.

Preclinical anti-tumor activity of antibody-targeted chemotherapy with CMC-544 (inotuzumab ozogamicin), a CD22-specific immunoconjugate of calicheamicin, compared with non-targeted combination chemotherapy with CVP or CHOP.

PURPOSE: CMC-544 (inotuzumab ozogamicin) is a CD22-specific immunoconjugate of calicheamicin currently being evaluated in patients with non-Hodgkin's B-cell lymphoma (BCL). CHOP and CVP represent untargeted combination chemotherapy comprised of cyclophosphamide, vincristine and prednisone with or without doxorubicin, commonly used in the treatment of NHL. Here, we describe anti-tumor efficacy of CMC-544, CHOP or CVP against human BCL xenografts. METHODS: In vitro, human BCLs were cultured with CMC-544 or individual constituents of CHOP for inhibition of their growth. In vivo, immunocompromised mice with established BCL xenografts were administered CHOP, CVP or CMC-544 to monitor their survival and BCL growth. RESULTS: In vitro, CMC-544 was more potent in causing growth inhibition of various BCL than cyclophosphamide, doxorubicin, vincristine or dexamethasone. In vivo, treatment with CHOP or CVP inhibited growth of BCL xenografts for up to 40 days after which BCL relapsed. Tumor growth inhibition by CMC-544 (>100 days) lasted longer than that by CHOP or CVP. BCL xenografts that relapsed after the treatment with CHOP or CVP were far less responsive to CHOP or CVP re-treatment but regressed upon subsequent treatment with CMC-544. CVP could be co-administered with suboptimal doses of CMC-544, while CHOP could be administered on alternant days with CMC-544 to cause enhanced regression of established BCL xenografts. CONCLUSION: Preclinically, CMC-544 provides greater therapeutic benefit than CVP or CHOP against BCL xenografts. CMC-544 may also be co-administered with standard chemotherapeutic regimens in the treatment of B-NHL for superior anti-tumor activity.

Determination of pharmacokinetic values of calicheamicin-antibody conjugates in mice by plasmon resonance analysis of small (5 microl) blood samples.

PURPOSE: The present study aims to establish a method that provides fast, precise and reproducible pharmacokinetic (PK) parameters of antibody-calicheamicin conjugates. The method should discriminate between PK of the antibody moiety and PK of the conjugated calicheamicin (CM). METHODS: The conjugates gemtuzumab ozogamicin (CMA-676, Mylotarg) or inotuzumab ozogamicin (CMC-544) were injected in the tail vein of nude mice. At regular time intervals, 5 mul whole blood samples were taken from the tail artery. Concentrations of conjugated CMA-676 or CMC-544 as well as concentrations of their respective antibody moiety were determined by sandwich plasmon resonance. This detection system measures changes in the plasma resonance angle caused by the interaction of macromolecules on biosensor chips. We determined as a first measure the binding of CMA-676 or CMC-544 to their respective antigens, CD33 or CD22. As a second measure we determined the amount of CM on the antigen-bound conjugates. This was done by determination of changes in plasma resonance angle after binding of an anti-CM antibody. RESULTS: Sandwich plasmon resonance allowed detection of both conjugates in blood of mice in a range of 100-1,000 ng/ml protein. Due to the precision of the sampling and detection methods, PK values of each conjugate were determined in individual mice. Calicheamicin bound to antibody was eliminated faster than the antibody alone. The presence of a CD22-expressing tumour in mice reduced the plasma levels of the CD22-targeting conjugate but not of the CD33-targeting one. CONCLUSIONS: Using small blood samples from a mouse, the sandwich plasmon resonance method provided PK-values of CM-conjugates and information about the stability of the linkage in vivo. Comparison between the PK-values of CM-conjugates in tumour-bearing and tumour-free mice suggested that retention of the conjugate in tumour tissue due to antigen targeting could be deduced from the plasma levels.

CD20-specific antibody-targeted chemotherapy of non-Hodgkin's B-cell lymphoma using calicheamicin-conjugated rituximab.

Tumor-targeted delivery of a potent cytotoxic agent, calicheamicin, using its immunoconjugates is a clinically validated therapeutic strategy. Rituximab is a human CD20-specific chimeric antibody extensively used in B-NHL therapy. We investigated whether conjugation to calicheamicin can improve the anti-tumor activity of rituximab against human B-cell lymphoma (BCL) xenografts in preclinical models. BCL cells were cultured with rituximab or its calicheamicin conjugates and their in vitro growth was monitored. BCL cells were injected s.c. to establish localized xenografts in nude mice or i.v. to establish disseminated BCL in severe combined immunodeficient (scid) mice. I.p. treatment with rituximab or its calicheamicin conjugates was initiated and its effect on s.c. BCL growth or survival of mice with disseminated BCL was monitored. Conjugation of calicheamicin to rituximab vastly enhanced its growth inhibitory activity against BCL in vitro. Conjugation to calicheamicin had no deleterious effect on the effector functional activity of rituximab. Calicheamicin conjugated to rituximab with an acid-labile linker exhibited greater anti-tumor activity against s.c. BCL xenografts and improved survival of mice with disseminated BCL over that of unconjugated rituximab. Anti-tumor activities of rituximab conjugated to calicheamicin via an acid-stable linker were similar to that of unconjugated rituximab. Superior anti-tumor efficacy exhibited by a calicheamicin immunoconjugate of rituximab with an acid-labile linker over that of rituximab demonstrates the therapeutic potential of CD20-specific antibody-targeted chemotherapy strategy in the treatment of B-NHL.

Biopharmaceutical drug discovery using novel protein scaffolds.

In recent years, biopharmaceutical drug products have become hugely successful. However, they are often complex molecules that are expensive to manufacture. Commercial needs for cost-effective therapies have therefore led to the development of novel protein scaffold technologies that are increasingly being used for biopharmaceutical drug discovery. Major new scaffolds include single-domain antibodies, small modular immunopharmaceuticals, tetranectins, AdNectins, A-domain proteins, lipocalins and ankyrin repeat proteins. These scaffolds offer low-cost alternatives to classical antibody therapeutic strategies and some have shown early clinical promise. Further progress in the field will permit the commercially successful development of sophisticated protein therapeutics against complex disease targets.

Tumoricidal effect of calicheamicin immuno-conjugates using a passive targeting strategy.

Calicheamicin is a potent chemotherapeutic with a low therapeutic index that requires targeting to tumor cells for its use in the clinic. To treat acute myeloid leukemia, calicheamicin has been conjugated to an antibody that recognizes CD33 (gemtuzumab ozogamicin). The application range of this 'active' targeting strategy is limited since it depends on specific antigen expression by tumor cells. This limitation could be reduced by using an antigen-independent 'passive targeting' strategy for calicheamicin. 'Passive targeting' relies on the dysfunctional vasculature of a neoplastic tumor that allows enhanced retention of macromolecules. We studied the efficacy of calicheamicin conjugated to various carrier molecules: i.e. immunoglobulin, albumin or PEGylated Fc fragments. In nude mice, a conjugate of anti-CD33 and calicheamicin accumulates in human tumor xenografts in the absence of detectable amounts of targeting antigen. Passive targeting provided sufficient accumulation of this conjugate to inhibit tumor growth of 10 different CD33-negative xenograft models. This efficacy depended on the use of an acid-labile linker between antibody and calicheamicin. Substitution of immunoglobulin as a carrier with either albumin or PEGylated Fc reduced or eliminated the efficacy of the conjugate. The results showed that using 'non-specific' immunoglobulin for passive targeting of calicheamicin might be an effective mode of cancer therapy.

Antitumor efficacy of a combination of CMC-544 (inotuzumab ozogamicin), a CD22-targeted cytotoxic immunoconjugate of calicheamicin, and rituximab against non-Hodgkin's B-cell lymphoma.

PURPOSE: CMC-544 is a CD22-targeted cytotoxic immunoconjugate, currently being evaluated in B-cell non-Hodgkin's lymphoma (B-NHL) patients. Rituximab is a CD20-targeted antibody commonly used in B-NHL therapy. Here, we describe antitumor efficacy of a combination of CMC-544 and rituximab against B-cell lymphoma (BCL) in preclinical models. EXPERIMENTAL DESIGN: BCLs were cultured in vitro with CMC-544, rituximab, or their combination. BCLs were injected either s.c. or i.v. to establish localized s.c. BCL in nude mice or disseminated BCL in severe combined immunodeficient mice, respectively. I.p. treatment with CMC-544 or rituximab was initiated at various times either alone or in combination and its effect on s.c. BCL growth or survival of mice with disseminated BCL was monitored. RESULTS: In vitro growth-inhibitory activity of CMC-544 combined with rituximab was additive. Rituximab but not CMC-544 exhibited effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Rituximab was less effective in inhibiting growth of established BCL xenografts than developing xenografts. In contrast, CMC-544 was equally effective against both developing and established BCL xenografts. Although CMC-544 and rituximab individually caused partial inhibition of the growth of BCL xenografts at suboptimal doses examined, their combination suppressed xenograft growth by >90%. In a disseminated BCL model, 60% of CMC-544-treated mice and 20% of rituximab-treated mice survived for 125 days. In contrast, 90% of mice treated with the combination of CMC-544 and rituximab survived for longer than 125 days. CONCLUSION: The demonstration of superior antitumor activity of a combination of CMC-544 and rituximab described here provides the preclinical basis for its clinical evaluation as a treatment option for B-NHL.

B-cell depletion inhibits arthritis in a collagen-induced arthritis (CIA) model, but does not adversely affect humoral responses in a respiratory syncytial virus (RSV) vaccination model.

We report the development of a mouse B cell-depleting immunoconjugate (anti-CD22 monoclonal antibody [mAb] conjugated to calicheamicin) and its in vivo use to characterize the kinetics of CD22+ B-cell depletion and reconstitution in murine primary and secondary lymphoid tissues. The effect of B-cell depletion was further studied in a murine collagen-induced arthritis (CIA) model and a respiratory syncytial virus (RSV) vaccination model. Our results show that (1) the immunoconjugate has B-cell-specific in vitro and in vivo cytotoxicity; (2) B-cell reconstitution starts in the bone marrow and spleen around day 30 after depletion and is completed in all tissues tested by day 50; (3) B-cell depletion inhibits the development of clinical and histologic arthritis in the CIA model; (4) depletion of type II collagen antibody levels is not necessary for clinical and histologic prevention of CIA; and (5) B-cell depletion does not adversely affect memory antibody responses after challenge nor clearance of infectious virus from lungs in the RSV vaccination model. These results demonstrate for the first time that only B-cell reduction but not type II collagen antibody levels correlate with the prevention of arthritis and represent key insights into the role of CD22-targeted B-cell depletion in mouse autoimmunity and vaccination models.

Antibody-targeted chemotherapy of B-cell lymphoma using calicheamicin conjugated to murine or humanized antibody against CD22.

Antibody-targeted chemotherapy with immunoconjugates of calicheamicin is a clinically validated strategy in cancer therapy. This study describes the selection of a murine anti-CD22 mAb, m5/44, as a targeting agent, its conjugation to a derivative of calicheamicin (CalichDM) via either acid-labile or acid-stable linkers, the antitumor activity of CalichDM conjugated to m5/44, and its subsequent humanization by CDR grafting. Murine IgG1 mAb m5/44 was selected based on its subnanomolar affinity for CD22 and ability to be internalized into B cells. CalichDM conjugated to m5/44 caused potent growth inhibition of CD22+ human B-cell lymphomas (BCLs) in vitro. The conjugate of m5/44 with an acid-labile linker was more potent than an acid-stable conjugate, a nonbinding conjugate with a similar acid-labile linker, or unconjugated CalichDMH in inhibiting BCL growth. CalichDM conjugated to m5/44 caused regression of established BCL xenografts in nude mice. In contrast, both unconjugated m5/44 and a nonbinding conjugate were ineffective against these xenografts. Based on the potent antitumor activity of m5/44-CalichDM conjugates, m5/44 was humanized by CDR grafting to create g5/44, an IgG4 anti-CD22 antibody. Both m5/44 and g5/44 bound CD22 with subnanomolar affinity. Competitive blocking with previously characterized murine anti-CD22 mAbs suggested that g5/44 recognizes epitope A located within the first N-terminal Ig-like domain of human CD22. Antitumor efficacy of CalichDM conjugated to g5/44 against BCL xenografts was more potent than its murine counterpart. Based on these results, a calicheamicin conjugate of g5/44, CMC-544, was selected for further development as a targeted chemotherapeutic agent for the treatment of B-cell malignancies.

Potent and specific antitumor efficacy of CMC-544, a CD22-targeted immunoconjugate of calicheamicin, against systemically disseminated B-cell lymphoma.

PURPOSE: CMC-544 is a CD22-targeted immunoconjugate of calicheamicin and exerts a potent cytotoxic effect against CD22+ B-cell lymphoma. This study evaluated antitumor efficacy of CMC-544 against systemically disseminated B-cell lymphoma. EXPERIMENTAL DESIGN: Scid mice received i.v. injections of CD22+ Ramos B-cell lymphoma cells for their systemic dissemination. CMC-544, G5/44, CD33-targeted CMA-676 (control conjugate) or rituximab were given i.p. 3, 9, 15, or 21 days after B-cell lymphoma dissemination. Diseased mice were monitored daily for hind-limb paralysis and death. Histopathological examination of CMC-544-treated and vehicle-treated diseased mice was also performed. RESULTS: Mice with disseminated B-cell lymphoma developed hind-limb paralysis within 35 days. When given up to 15 days after B-cell lymphoma dissemination, CMC-544 extended survival of the diseased mice to >100 days, and these mice were considered cured. CMC-544 was efficacious when given during both the early initiation phase and the late established phase of the disease. A single dose of CMC-544 was effective in delaying the occurrence of hind-limb paralysis. In contrast, neither CMA-676 nor unconjugated G5/44 was effective. Rituximab was effective when given early in the disease process but not when the disease was established. Histopathological analysis revealed B-cell lymphoma infiltration in brain, spinal cord, bone marrow, and kidney in vehicle-treated but not in CMC-544-treated diseased mice. Consistent with its efficacy against the disseminated B-cell lymphoma, CMC-544 also caused regression of established Ramos B-cell lymphoma xenografts in scid mice. CONCLUSIONS: CMC-544 confers strong therapeutic activity against systemic disseminated B-cell lymphoma and protects mice from hind-limb paralysis and death. These results support clinical evaluation of CMC-544 in the treatment of CD22+ lymphoid malignancies.

Tumour-targeted chemotherapy with immunoconjugates of calicheamicin.

Antibody-targeted chemotherapy is a therapeutic strategy in cancer therapy that involves a monoclonal antibody specific for a tumour-associated antigen, covalently linked via a suitable linker to a potent cytotoxic agent. Tumour-targeted delivery of a cytotoxic agent in the form of an immunoconjugate is expected to improve its antitumour activity and safety. Calicheamicin is a cytotoxic natural product isolated from Micromonospora echinospora that is at least 1000-fold more potent than conventional cytotoxic chemotherapeutics. Calicheamicin binds DNA in the minor groove and causes double-strand DNA breaks, leading to cell death. Immunoconjugates of calicheamicin targeted against tumour-associated antigens exhibit tumour-specific cytotoxic effects and cause regression of established human tumour xenografts in nude mice. Gemtuzumab ozogamicin is the first clinically validated cytotoxic immunoconjugate in which a humanised anti-CD33 antibody is linked to a derivative of calicheamicin. Gemtuzumab ozogamicin is indicated for the treatment of elderly patients with relapsed acute myeloid leukaemia. A similar conjugate, inotuzumab ozogamicin, is being evaluated at present in Phase I clinical trials in patients with non-Hodgkin's lymphoma. A number of tumour-targeted immunoconjugates of calicheamicin are being explored preclinically at present for their therapeutic applications.

Antibody-targeted chemotherapy with the calicheamicin conjugate hu3S193-N-acetyl gamma calicheamicin dimethyl hydrazide targets Lewisy and eliminates Lewisy-positive human carcinoma cells and xenografts.

PURPOSE: Linking a cytotoxic anticancer drug to an antibody that recognizes a tumor-associated antigen can improve the therapeutic index of the drug. We asked whether a conjugate of the cytotoxic antibiotic N-acetyl gamma calicheamicin dimethyl hydrazide (CalichDMH) and an antibody recognizing Lewis(y) (Le(y)) antigen could eliminate carcinomas that express Le(y). Because Le(y) is highly expressed on carcinomas of colon, breast, lung, ovary, and prostate, a CalichDMH conjugate targeting Le(y) could provide a treatment option for various cancers. EXPERIMENTAL DESIGN: The humanized anti-Le(y) antibody hu3S193 was conjugated to CalichDMH via the bifunctional AcBut linker. Selectivity and avidity of the conjugate (hu3S193-CalichDMH) for Le(y)-BSA or Le(y+) cells was tested by BIAcore or flow cytometry. Cytotoxicity of hu3S193-CalichDMH was compared with toxicity of a control conjugate on monolayers of Le(y+) and Le(y-) carcinoma cells. Inhibition of tumor growth by hu3S193-CalichDMH was assessed on three types of s.c. xenografts. RESULTS: Hu3S193-CalichDMH had similar selectivity as hu3S193. The conjugate had lower affinity for Le(y)-BSA but not for Le(y+) cells. When tested on monolayers of human Le(y+) carcinoma cells, hu3S193-CalichDMH was more cytotoxic than a control conjugate. This difference in efficacy was not noted on Le(y-) cells. Efficacy of hu3S193-CalichDMH depended on the expression of Le(y) and on the sensitivity of the cells to CalichDMH. In vivo, hu3S193-CalichDMH inhibited growth of xenografted human gastric (N87), colon (LOVO), and prostate carcinomas (LNCaP). When used against N87 xenografts, hu3S193-CalichDMH arrested tumor growth for at least 100 days. CONCLUSION: Hu3S193-CalichDMH can specifically eliminate Le(y+) tumors. These results support development of this conjugate for treatment of carcinomas.

Antibody-targeted chemotherapy with CMC-544: a CD22-targeted immunoconjugate of calicheamicin for the treatment of B-lymphoid malignancies.

Antibody-targeted chemotherapy with gemtuzumab ozogamicin (CMA-676, a CD33-targeted immunoconjugate of N-acetyl-gamma-calicheamicin dimethyl hydrazide [CalichDMH], a potent DNA-binding cytotoxic antitumor antibiotic) is a clinically validated therapeutic option for patients with acute myeloid leukemia (AML). Here, we describe the preclinical profile of another immunoconjugate of CalichDMH, CMC-544, targeted to CD22 expressed by B-lymphoid malignancies. CMC-544 comprises a humanized IgG4 anti-CD22 monoclonal antibody (mAb), G5/44, covalently linked to CalichDMH via an acid-labile 4-(4'-acetylphenoxy) butanoic acid (AcBut) linker. Both CMC-544 and unconjugated G5/44 bound human CD22 with subnanomolar affinity. CMC-544, but not unconjugated G5/44, exerted potent cytotoxicity against CD22+ B-cell lymphoma (BCL) cell lines (inhibitory concentration of 50%: 6-600 pM CalichDMH). CMC-544 caused a potent inhibition of growth of small but established BCL xenografts leading to cures (therapeutic index > 10). CMC-544 prevented the establishment of BCL xenografts and also caused regression of large BCLs (> 1.5 g tumor mass). In contrast, unconjugated CalichDMH, unconjugated G5/44, and an isotype-matched control conjugate, CMA-676, were ineffective against these BCL xenografts. Thus, CD22-targeted delivery of CalichDMH is a potent and effective preclinical therapeutic strategy for BCLs. The strong antitumor profile of CMC-544 supports its clinical evaluation as a treatment option for B-lymphoid malignancies.

Antibody-targeted chemotherapy with immunoconjugates of calicheamicin.

Targeted delivery of cytotoxic agents to tumours is believed to improve both their anti-tumour efficacy and their safety. Antibodies specific for tumour-associated antigens have been used to deliver cytotoxic agents to tumour cells. Calicheamicin is a potent cytotoxic agent that causes double-strand DNA breaks, resulting in cell death. When conjugated to monoclonal antibodies specific for tumour-associated antigens, calicheamicin exerts strong antigen-specific anti-tumour effects against human tumour xenografts in preclinical models. Antibody-targeted chemotherapy with immunoconjugates of calicheamicin, exemplified by gemtuzumab ozogamicin (Mylotarg), is a clinically validated therapeutic strategy for the treatment of human cancer.

Reduced expression of EphrinA1 (EFNA1) inhibits three-dimensional growth of HT29 colon carcinoma cells.

Ephrin A1 (EFNA1) is a GPI-anchored ligand that preferentially binds to the receptor tyrosine kinase, EphA2. EphA2 is over-expressed in malignant melanocytes and in prostate carcinoma cells. Whether activation of EphA2 by EFNA1 is involved in aberrant growth or differentiation of cancer cells is currently not known. We studied the effect of reducing EFNA1 on the growth of a colon carcinoma cell line (HT29). HT29 cells were transfected with EFNA1 antisense yielding clones that expressed less than 25% of EFNA1 found in vector controls. EFNA1-antisense transfectants grew slower than controls when cultured as three-dimensional spheroids. When grown as monolayers, the transfectants had a similar doubling time of the vector controls. These results indicated that autocrine stimulation of EphA2 by EFNA1 could trigger an indirect growth signal by overcoming 'contact inhibition'. Following addition of EFNA1-Fc to HT29 cells, tyrosine hyperphosphorylation of EphA2, E-cadherin, and beta-catenin were observed. Because the function of E-cadherin is associated with contact inhibition of HT29 cells, phosphorylation of E-cadherin and beta-catenin by activation of EphA1 is one possible mechanism by which HT29 cells alleviate contact inhibition.